JP2023521800A - In vitro production and purification of therapeutic mRNA - Google Patents

Abstract

The present invention includes novel systems, methods, and compositions for the in vitro production of polynucleotides, particularly mRNA for use in therapeutic applications. [Selection figure] None

Description

This international PCT application claims the benefit of and priority to US Provisional Application No. 63/011133, filed April 16, 2020, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on April 16, 2021, is named "90125-00181-Sequence-Listing-AF.txt" and is 26.9 Kbytes in size.

The present invention relates generally to the field of in vitro production of polynucleotides, and in particular to the production of mRNA for use in therapeutic applications.

Messenger RNA (mRNA) is a template molecule that is transcribed from a cell's DNA and translated into an amino acid sequence, ie, a protein, in the ribosomes within the cells of an organism. To control the level of expression of the encoded protein, the mRNA has untranslated regions (UTRs) flanking the actual open reading frame (ORF) that contains the genetic information encoding the amino acid sequence. Such UTRs are termed 5'-UTR and 3'-UTR, respectively, and are the stretches of mRNA located before the start codon and after the stop codon. In addition, mRNA contains a poly(A) tail (poly(A) strand) region, a long sequence of adenine nucleotides that facilitates transport of the mRNA from the nucleus and provides some protection from degradation of the mRNA. Recent advances in science and technology have made mRNA a promising candidate for a variety of uses, including diagnostic applications and therapeutic products such as vaccines.

Due to the growing demand, such as the recent COVID-19 pandemic, from a healthcare community that not only enables personalized medicine, but also emergency response in epidemic crisis situations. , many approaches have been developed for large-scale production of mRNA. Most current methods utilize fermentation to synthesize mRNA from self-replicating DNA templates in culture, and then volatile organic solvents to isolate total RNA as a raw material. These processes are costly, hazardous, and generate a series of hazardous waste streams that must be mediated, while production rates depend on the performance of the producing strain and the variety of tRNA, rRNA, and host Much depends on the ability to remove impurities from the mRNA.

As can be seen, homogeneous and pure mRNA suitable for therapeutic use while not requiring volatile organic solvents, not generating a hazardous waste set, and significantly lower cost than its fermentation-based counterparts. There is a long felt need for an efficient in vitro mRNA manufacturing process that produces .

One aspect of the invention includes novel in vitro methods for the production of polynucleotides and mRNA that may be specifically targeted for one or more diagnostic or therapeutic uses. In one preferred aspect, the invention comprises a fully recombinant, stable, reliable and functional in vitro system for batch or continuous flow production of polynucleotides and preferably RNA. In this preferred embodiment, the present improved in vitro system utilizes components involved in the transcription of polynucleotides to create an in vitro environment configured to mimic the production of polynucleotides that occurs in vivo. can.

Another aspect of the invention includes novel in vitro methods for the production of mRNA. In this embodiment, an in vitro bioreactor (and preferably a batch bioreactor) or continuous flow bioreactor comprises an isolated RNA polymerase (RNAP) and a nucleotide template (and preferably a linear non-self-replicating DNA template). ), together with multiple nucleotide triphosphates (NTPs) (which can be incorporated into mRNA molecules synthesized through the action of RNAP), and an energy source (e.g., Koglin and Humbert, PCT Application No. PCT/US2018/012121). (the descriptions, figures, examples, sequences, and claims of which are hereby incorporated by reference in their entirety). can be configured. In preferred embodiments, the synthesized mRNA can then be purified and used for a variety of downstream purposes, including diagnostic or therapeutic applications such as vaccines directed against selected target pathogens.

In preferred embodiments, the production of macromolecules using the recombinant cell-free systems of the invention can be accomplished in bioreactor systems. As used herein, a "bioreactor" is configured to maintain an environment conducive to the production of macromolecules, and preferably polynucleotide macromolecules, even more preferably mRNA transcribed in vitro from a DNA template. It may be any form of sealing device provided. A bioreactor can be configured to operate on a batch, continuous, or semi-continuous basis, eg, with a feed or feed solution. In one aspect, the invention can further include a bioreactor configured to produce mRNA. In this aspect, the invention may be particularly suitable for operation with continuous-flow bioreactor systems, which comprise one or more hollow continuous-flow conduits (e.g., made of fibrous material in fluid communication with an external bioreactor compartment). ). In this aspect, the hollow continuous flow conduit is formed as an exchange medium for in vitro transcripts or polynucleotides (and preferably mRNA).

Another aspect of the invention includes the synthetic biological production of polynucleotides (and preferably mRNAs via various reaction types). In one embodiment, a batch reaction can be used to produce mRNA in vitro. In this preferred embodiment, the isolated RNAP, DNA template, and NTPs can all be combined into a batch-fed reaction chamber and incubated until the reactants are consumed. To circumvent the scaling limitations of batch reactions, in another preferred embodiment, continuous flow bioreactors can be used to produce mRNA in vitro. A continuous flow bioreactor can be configured to operate continuously such that new input material can be injected while producing large amounts of mRNA that can be output during or after the reaction process.

Another aspect of the invention may include novel systems and methods for the isolation and purification of polynucleotides (and, preferably, mRNA produced in the in vitro production systems of the invention). In a preferred embodiment, the reaction output containing synthesized mRNA is purified from the mRNA output without the use of hazardous organic solvents or expensive single-use purification kits for the reaction mixture components i.e. RNAP, template DNA, free NTPs. , and buffers can be removed sequentially. In one embodiment, the present invention can utilize a purification column cascade, where reaction material from either batch or continuous flow bioreactors is subjected to protein affinity resin followed by DNA affinity resin. washed or vice versa. This step can remove all protein and DNA template reaction components, except for unreacted free nucleotides. Free NTPs and buffer can be removed by alcohol precipitation, leaving only the isolated precipitated mRNA material. The mRNA can then be resolubilized for desired downstream use or dried for long term storage or reduced volume delivery.

Another aspect of the invention is pharmaceuticals for novel mRNA-based vaccines directed against target pathogens, such as COVID-19, produced by one or more of the in vitro mRNA production methods described herein. therapeutic compositions and their use to treat a subject in need thereof.

Another aspect of the invention can include novel systems, methods, and compositions for multi-step in vitro production of mRNA. In one preferred aspect, mRNA production from a DNA template is separated from poly(A) tail addition, or poly(A) tailing. For example, in a first bioreactor (and preferably a continuous flow bioreactor of the invention), mRNA is produced from a DNA template. The production of this first stage production step may or may not be coupled with the addition of a 5' cap to the mRNA transcript. In a second step, the mRNA transcript can be introduced into a second bioreactor (and preferably a continuous flow bioreactor of the invention) and further modified to include a poly(A) tail. Optionally, the mRNA can be modified to include a 5' prime cap, assuming it was not included in the first bioreactor, as described above.

Another aspect of the invention can include novel systems, methods, and compositions for multi-step in vitro production of mRNA. In one preferred aspect, mRNA production from a DNA template is separated from poly(A) tail addition, or poly(A) tailing. For example, in a first bioreactor (and preferably a continuous flow bioreactor of the invention), mRNA is produced from a DNA template. The production of this first stage production step may or may not be coupled to the addition of a 5' cap to the mRNA transcript. In a second step, the mRNA transcript can be introduced into a second bioreactor (and preferably a continuous flow bioreactor of the invention) and further modified to include a poly(A) tail. Optionally, the mRNA can be modified to include a 5' prime cap, assuming it was not included in the first bioreactor, as described above. This multi-step in vitro mRNA production has several advantages. For example, post-transcriptional poly(A) tail generation is separated from RNA synthesis, allowing the operator to limit unwanted synthesis of double-stranded RNA (dsRNA). The multi-step production system of the present invention also reduces manual handling of mRNA, further reducing the risk of shearing. Finally, the multi-step production system of the present invention allows one-step isolation/purification of mRNA, eg, via only poly(A) tailed RNA binding to poly dT resin.

Another aspect of the invention may include novel systems, methods, and compositions for multi-step in vitro production of mRNA with a Cap1 enzymatic processing system. In one preferred aspect, the Cap0/Cap1 enzymatic treatment system can act as a checkpoint that recognizes Cap0 on RNA, followed by enzymatic methylation to generate Cap1 followed by poly(A) tails. can. This choice of poly(A) tail ensures that the RNA is also fully capped during production, resulting in improved yields of fully functional mRNA.

Another aspect of the invention may involve multi-step in vitro production of novel mRNAs in which mRNA synthesis and poly(A) tailing are separated. In this aspect, a reaction mixture having a DNA template (e.g., linearized plasmid, PCR product, amine-functionalized surface-tethered PCR product), a first amount of RNA polymerase, and a reaction buffer is combined with the batch or continuous method of the invention. Introduce into a first bioreactor, which may comprise a flow bioreactor or other suitable bioreactor (eg, a hollow fiber reactor as described herein). In a preferred embodiment, the reaction mixture is introduced into and passed through the internal reaction cell of the first bioreactor. This internal reaction cell consists of a feed solution containing nucleotide NTPs, a reaction buffer which may contain the necessary components to catalyze and drive mRNA synthesis as described herein, and the hydration of pyrophosphate to inorganic phosphate. It may be in fluid communication with a supply chamber holding pyrophosphatase that catalyzes degradation. In this embodiment, the feed solution can be circulated in countercurrent flow compared to the reaction mixture. In this embodiment, the feed chamber and reaction cell can be configured to include the continuous flow aspects outlined herein.

As described above, the compartmentalization and countercurrent flow of the feed solution and reaction mixture ensures that free NTs from the feed solution, as they pass through the feed chamber, are drawn into the internal compartment of the reaction cell where they react with the constituents of the reaction mixture. Generate the gradients so that In this embodiment, RNAP can associate with a DNA template having a target sequence encoding a target mRNA and enzymatically catalyze the incorporation of NT into target mRNA nucleotides.

The reaction mixture from the internal reaction cell, after undergoing one or more reaction cycles, can be extracted and introduced into the mixing cell, where the newly synthesized mRNA is mixed with the buffer solution, e.g. (and preferably in a high salt buffer). Dilution and addition of high salt buffer produces a highly concentrated RNA solution, inactivates RNAP, and further adjusts buffer conditions to reduce poly(A) during the second stage poly(A) tailing step. Allows enzymatic activity of the polymerase.

In preferred embodiments, enzymes for 5' capping and poly(A) tailing of mRNA transcripts may also be provided in the dilution buffer. This new reaction mixture is then transferred to the reaction cell of a second hollow fiber reactor with a feed chamber containing additional components such as nucleotide triphosphates (e.g., ATP and GTP) and S-adenosylmethionine (SAM). introduced into As described above, these components pass through a porous barrier, such as the hollow fiber barrier (34) described herein, into the reaction cell and undergo 5' capping and poly(A) tailing of mRNA transcripts. can be placed in a feed chamber that forms a gradient in the reaction cell to act as a substrate for the In this configuration, the mRNA is capped and poly(A) tailed and the reaction mixture from the reaction cell of the second hollow fiber reactor is diluted in high salt buffer to form a poly(A) tailed RNA poly(A) tail. Allows slow binding to dT resin. Other components pass over the resin and are collected. In certain embodiments, the recombinant enzyme from the reaction mixture is captured by the affinity resin on the column and then washed on the poly dT resin. The reaction mixture was washed thoroughly on poly dT resin, and after the poly(A)-modified RNA was bound to the resin, it was washed with flowing high salt buffer, and after washing, the distilled poly(A)-modified RNA was washed with distilled water. Release from the resin. The final product of the multi-step in vitro production system is concentrated capped and poly(A)-tailed RNA in distilled water, which can be further processed such as encapsulation into lipid nanoparticles (LNPs) for therapeutic applications. It can be subjected to further processing.

Further aspects of the present technology will become apparent from the following specification, drawings, and claims.

Aspects, features, and advantages of the present disclosure will be better understood from the following detailed description in conjunction with the accompanying drawings. All of these are provided merely as examples and are not intended to limit embodiments of the present disclosure.

1 shows a system schematic of a continuous-flow mRNA production system, in one embodiment. 1 shows a schematic diagram of a system of an mRNA batch production system in one embodiment thereof. FIG. 1 shows a schematic diagram of the mRNA purification and isolation system in one embodiment thereof. An exemplary 1.0% agarose gel demonstrating mRNA production reactions performed on March 30, 2020, April 1, 2020, and April 3, 2020 is shown. A marker ladder (purchased from New England Biolabs (NEB)) is loaded in lane 1. A total of 10 ul of unpurified reactions (bulk) and purified reactions from March 30, 2020 are loaded in lanes 2 and 3 on the gel. A total of 10 ul of unpurified reactions (bulk) and purified reactions from 04/01/2020 are loaded in lanes 4 and 5 on the gel. Lanes 6-11 show 10 ul samples taken from the purification process outlined herein. Lane 6 is the unpurified reaction (bulk), lane 7 is the material after applying the reaction mixture to the protein affinity column, lane 8 is the product after applying the reaction mixture to the DNA affinity column. Lane 9 shows the reaction mixture after washing via alcohol precipitation and then subsequent dissolution in sterile water; Lane 10 shows the precipitate and isolated product after sterile filtration; 10 shows the precipitated and isolated mRNA product after being purified and optionally concentrated through an RNA purification column (provided by NEB). Gels are visualized by illumination at 365 nm on a UV table. 1 shows a system schematic of a multi-step mRNA production system, in one embodiment thereof. 1 shows a system schematic of a multi-step mRNA production system utilizing multiple hollow fiber bioreactors, in one embodiment thereof. FIG. Bioanalyzer analysis. Three different mRNA product samples were capped and tailed using the system outlined in Figure 5, and pre/post samples were run on the Agilent Bioanalyzer 2100 using the Agilent RNA 6000 Nano kit according to the manufacturer's instructions. run on the instrument. Analysis of the samples shows an apparent visual size shift due to tailing and a minor population of untailed mRNA. 200 ng of raw mRNA versus capped and tailed mRNA on Bioanalyzer 2100. Expected column size: m001 about 1650nt, m002 about 1675nt, m003 about 2600nt. Tailing adds about 250-300 nt. Denaturing agarose gel analysis. In vitro transcribed large (11000 nt) mRNA was produced according to the system described in Figure 5, purified and analyzed on a denaturing agarose gel. Long mRNA samples were analyzed by dissolving 1% agarose, using 2.5% formaldehyde and 0.01% gel red, and pouring denaturing gels. A 1 μg mRNA sample was run alongside a commercially available RNA ladder (RiboRuler HR). Denaturing gels prevent most secondary structure formation, but a small smear of various mRNA populations will still be visible. Successful production of such large mRNA species demonstrates the feasibility of the inventive system described herein.

Referring generally to FIG. 1, in one embodiment, the technology of the present invention may comprise a novel continuous flow bioreactor (1) configured for scaled in vitro production of polynucleotides (particularly mRNA). . Generally, referring to FIG. 1, a continuous flow conduit (3) may pass through a continuous flow reaction chamber (2). In this embodiment, the continuous flow conduit (3) can continuously recycle the feed solution. The feed solution may contain one or more of the following components:
- a first amount of isolated NTPs,
- an amount of reaction buffer,
- optionally a component of the inorganic polyphosphate energy regeneration system described in PCT/US2018/012121, and - optionally one or more cofactors for the production of mRNA polynucleotides.

In this embodiment, the continuous flow reaction chamber (2) can hold the reaction mixture for the production of polynucleotides (particularly mRNA). This input reaction mixture may contain one or more of the following components:
- a first amount of isolated RNAP enzyme,
- a first amount of DNA template,
- an amount of reaction buffer,
- Optionally, an initial amount of isolated NTPs.

In particular, continuous flow conduit (3) may be in fluid communication with continuous flow reaction chamber (2). Specifically, as shown in Figure 1, the continuous flow conduit (3) allows one or more components of the feed solution to pass from multiple conduit openings (4) into the continuous flow conduit (3). It may be in fluid communication with the continuous flow reaction chamber (2) through a plurality of conduit openings (4) configured to. In one preferred embodiment, the continuous flow conduit (3) may comprise a continuous flow conduit (3) made from hollow fibers. In this embodiment, the continuous flow conduit (3) can be made from a MWCO PES membrane with pores between the size of 5 kDa or 20 kDa. This fiber membrane can be further treated to reduce protein and nucleotide binding. This treatment involves depositing RNA-free acetylated BSA on the outside of the conduit, and purified RNA polymerase can be placed inside the conduit where mRNA can be produced. In this configuration, a plurality of conduit openings (4) draws liberated NT from the feed solution as it passes through the continuous flow reaction chamber (2) into the internal compartment of the continuous flow reaction chamber (2) where the reaction mixture Generate a gradient so that it can react with the components of In this embodiment, RNAP associates with a DNA template (and preferably a linear DNA template) having a target sequence encoding target mRNA (14) and enzymatically induces incorporation of NTPs into target mRNA (14) nucleotides. can catalyze

In certain embodiments, the target mRNA (14) can be configured to produce a three-dimensional structural configuration of a hairpin configuration (e.g., a dsRNA configuration), which requires induction of the RNA interference pathway. Although it can be used to induce it in a subject, in further embodiments the target mRNA (14) can be used as a pharmaceutical composition. For example, in one embodiment, target mRNA (14) may encode an antigenic peptide (eg, against a viral protein). In this embodiment, mRNA can be administered to a subject in need thereof and translated to form antigenic proteins, which can then elicit an immune response.

For example, in one preferred embodiment, the continuous flow bioreactor (1) produces mRNA encoding one or more antigenic peptides directed to elicit an immune response directed against the COVID-19 coronavirus. can be configured to produce in a subject in need of More specifically, in this embodiment, the continuous flow bioreactor (1) is described in U.S. Application No. 62/992,072 by Koglin and Humbert (description, figures, sequences and constructs incorporated herein by reference). (specifically incorporated herein by reference).

In some embodiments, at least one coding region of the mRNA produced according to the present invention comprises or consists of a COVID-19 coronavirus protein or fragment or variant thereof, at least 2, 3, 4, It encodes 5, 6, 7, 8 or more antigenic peptides or proteins. More preferably, at least one coding region comprises the spike protein subunit 1 (S1) of the COVID-19 coronavirus, ii) the receptor binding motif (RBM) of S1, and ii) the nucleocapsid protein (NCP), and fragments thereof or variants, or fragments or variants of any one of these proteins, which may be further conjugated with a signal peptide, preferably an IgE signal peptide (which incorporates SEQ ID NO: 9) encodes at least 2, 3, 4, 5, 6, 7, 8 or more antigenic peptides or proteins. Even more preferably, at least one coding region is selected from the group consisting of integrated SEQ ID NOS: 1-6In, or fragments or variants of any one of these amino acid sequences, at least two, three , encodes a sequence of 4, 5, 6, 7, 8 or more amino acids. In particular, sequences that are incorporated are identified by the "In" designation.

Referring again to FIG. 1, the components of the reaction mixture can be loaded into the internal compartments of the continuous flow reaction chamber (2) prior to initiation of the reaction or replenished during operation as needed. A feed solution can also be loaded into the continuous flow conduit (3) prior to initiation of the reaction. For example, as shown in Figure 1, the feed solution is delivered to an input reservoir (5) coupled with an input valve (7) configured to allow real-time injection of the feed solution into the continuous flow conduit (3). to the continuous flow conduit (3). In certain embodiments, addition of the feed solution may be accomplished manually, and in alternate embodiments the process may be automated. In this latter embodiment, the feed solution is supplied based on a predetermined schedule or a predetermined threshold (e.g., the concentration of NTPs in the feed solution, the concentration of mRNA synthesized in the continuous flow reaction chamber (2), or the reaction can be added to the system based on another parameter, such as the energy consumption of Such parameters are measured by one or more sensors in communication with a computer system configured to execute a computer executable program in response to changes in one or more parameters as outlined herein. obtain.

In particular, after initiation of the reaction in the internal compartment of the continuous-flow reaction chamber (2), the incorporation of NTPs into newly synthesized mRNA, among other factors, allows fresh NTPs to flow continuously into the continuous-flow conduit (3). can be provided in a timely manner. Such quantitation is, for example, from the group consisting of nucleotide triphosphates, such as adenine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). It allows more efficient use of reactive enzymes and energy usage in the form of energy hydrolysis of one or more selected nucleotide triphosphates).

The continuous flow bioreactor (1) of the present invention may generally be further configured to include an inorganic polyphosphate energy regeneration system, including a cellular adenosine triphosphate (ATP) energy regeneration system. In this embodiment, the continuous flow reaction chamber (2) comprises:
- an amount of an isolated adenosyl kinase enzyme (and Gst AdK, preferably from a thermophilic bacterium);
- an amount of an isolated polyphosphate kinase enzyme (Taq PPK from thermophilic bacteria),
- an amount of inorganic polyphosphate (PPi) from thermophilic bacteria;
- an amount of adenosine monophosphate (AMP);

In this embodiment, the adenosyl kinase (AdK) and polyphosphate kinase (PPK) enzymes work synergistically to regenerate cellular ATP energy from PPi and AMP. More specifically, in another preferred embodiment, isolated and purified Gst AdK ( SEQ ID NO: 8In (incorporated herein by reference)) and/or Taq PPK (SEQ ID NO: 11In of the '121 application (incorporated herein by reference)), together with an amount of inorganic polyphosphate, can be added to this cell-free expression system. In one embodiment, this amount of inorganic polyphosphate may comprise the optimal polyphosphate concentration range. In this preferred embodiment, such optimal polyphosphate concentration range is generally defined as the concentration of inorganic polyphosphate (PPi) that keeps the equilibrium of the reaction stable. In this preferred embodiment, the optimal polyphosphate concentration range may be about 0.2-2 mg/ml PPi.

As mentioned above, PPK can synthesize ADP from polyphosphate and AMP. In this preferred embodiment, the combined action of Gst AdK and PPK removes adenosine diphosphate (ADP) from the system by converting two ADPs into one ATP and one adenosine monophosphate (AMP). can.

This reaction may be fast enough to drive the equilibrium reaction of PPK towards the production of ADP.

In this system, the presence of higher concentrations of AMP may drive the response of Taq PPK further towards ADP.

A portion of the reaction mixture and the mRNA output (9) containing newly synthesized mRNA in the continuous flow reaction chamber (2) can be extracted for further modification. Referring again to Figure 1, the mRNA output (9) is an output reservoir coupled with an output valve (8) configured to allow extraction of the mRNA output (9) from the continuous flow reaction chamber (2). Continuous flow to (6) can be extracted into reaction chamber (2). In certain embodiments, extraction of mRNA output (9) can be accomplished manually, but in alternative embodiments the process can be automated. In this latter embodiment, the mRNA output (9) is determined based on a predetermined schedule or at predetermined thresholds (e.g. concentration of NTPs in the feed solution, concentration of mRNA synthesized in the continuous flow reaction chamber (2) , or another parameter such as the energy consumption of the reaction). Such parameters are measured by one or more sensors in communication with a computer system configured to execute a computer executable program in response to changes in one or more parameters as outlined herein. obtain.

Referring now generally to FIG. 2, in one embodiment the technology of the present invention comprises a novel batch-fed reaction chamber (10) configured for scaled in vitro production of polynucleotides (particularly mRNA). can include In this embodiment, the batch-fed reaction chamber (10) may hold reaction mixtures for the production of polynucleotides (particularly mRNA). This input reaction mixture may contain one or more of the following components:
- a first amount of isolated RNAP enzyme,
- a first amount of DNA template,
- an amount of reaction buffer,
- a first amount of isolated NTPs,
- optionally a component of the inorganic polyphosphate energy regeneration system described in PCT/US2018/012121, and - optionally one or more cofactors for the production of mRNA polynucleotides.

In this configuration, the free RNAP is associated with a DNA template (and preferably a linear DNA template) having a target sequence encoding target mRNA (14), and the target mRNA (14) within the batch-fed reaction chamber (10). Incorporation of NTPs into nucleotides can be enzymatically catalyzed. Referring again to Figure 1, one or more components of the reaction mixture may enter the batch feed reaction chamber (10) through the input reservoir (5).

In certain embodiments, the target mRNA (14) produced in the batch-fed reaction chamber (10) can be configured to produce a three-dimensional structural configuration (e.g., a dsRNA configuration) in a hairpin configuration, which can be used to induce induction of the RNA interference pathway in a subject in need thereof, but in a further embodiment the target mRNA (14) can be used as a pharmaceutical composition. For example, in one embodiment, target mRNA (14) may encode an antigenic peptide (eg, against a viral protein). In this embodiment, mRNA can be administered to a subject in need thereof and translated to form antigenic proteins, which can then elicit an immune response.

For example, in one preferred embodiment, the batch-fed reaction chamber (10) encodes one or more antigenic peptides directed to elicit an immune response to the COVID-19 coronavirus in a subject in need thereof. can be configured to produce an mRNA that More specifically, in this embodiment, the continuous flow bioreactor (1) is described in U.S. Application No. 62/992,072 by Koglin and Humbert (description, figures, sequences and constructs incorporated herein by reference). (specifically incorporated herein by reference).

In some embodiments, at least one coding region of the mRNA produced according to the present invention comprises or consists of a COVID-19 coronavirus protein or fragment or variant thereof, at least 2, 3, 4, It encodes 5, 6, 7, 8 or more antigenic peptides or proteins. More preferably, at least one coding region comprises the spike protein subunit 1 (S1) of the COVID-19 coronavirus, ii) the receptor binding motif (RBM) of S1, and ii) the nucleocapsid protein (NCP), and fragments thereof or variants, or fragments or variants of any one of these proteins, which may be further conjugated with a signal peptide, preferably an IgE signal peptide (which incorporates SEQ ID NO: 9) encodes at least 2, 3, 4, 5, 6, 7, 8 or more antigenic peptides or proteins. Even more preferably, at least one coding region is selected from the group consisting of integrated SEQ ID NOS: 1-6In, or fragments or variants of any one of these amino acid sequences, at least two, three , encodes a sequence of 4, 5, 6, 7, 8 or more amino acids.

In particular, after initiation of the reaction in the batch-fed reaction chamber (10), once the NTPs are incorporated into the newly synthesized mRNA, the synthesis reaction is allowed to reach a threshold, such as a predetermined time or concentration of the newly synthesized mRNA. is satisfied or the enzyme or energy source of the reaction mixture is exhausted. As noted above, in one embodiment, the batch-fed reaction chamber (10) is a form of energetic hydrolysis of nucleotide triphosphates (e.g., one or more nucleotide triphosphates selected from the group consisting of) Energy sources may include: adenine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). Finally, the batch-fed reaction chamber (10) of the present invention may be further configured to include the inorganic polyphosphate energy recovery system outlined above.

The technology of the present invention provides a system and Further comprising a method. Referring generally to Figure 3, in one embodiment, mRNA output (9) can be passed through a protein affinity column (11) configured to capture the protein fraction (15) of mRNA output (9). , which may contain free RNAP or other proteins that may be present in the mRNA output (9). In embodiments, the captured RNAP can be eluted from the protein affinity column (11) and reused in subsequent in vitro mRNA production reactions.

As further shown in FIG. 3, after removing the protein fraction (15), the mRNA output (9) is transformed into a DNA fraction of the mRNA output (9), which may contain free DNA templates that may be present in the mRNA output (9). It can pass through a DNA affinity column (12) configured to capture the minute (16).

Again, as shown in FIG. 3, after removal of the protein fraction (15) and DNA fraction (16), the mRNA output (9) is reduced by the NTP fraction (17) and excess buffer or mRNA output ( It may pass through a nucleotide precipitator (13) configured to remove other components of 9). In a preferred embodiment, the free NTP fraction (17) is separated and removed from the target mRNA (14) via one or more rounds of alcohol precipitation and washing to render the target mRNA present in the mRNA output (9). (14) can be extracted. The finally isolated target mRNA (14) can be further purified and optionally resolubilized for desired downstream uses or dried for long term storage or reduced volume delivery. As above, the order of protein affinity column (11) and DNA affinity column (12) is first to remove the DNA fraction (16), second to remove the protein fraction (15), Also, vice versa can be switched to be removed as well. Such a sequence of operations is also applicable to the removal of the free NTP fraction (17).

The present invention can include systems, methods, and compositions for hollow fiber bioreactors (20) that can be configured for multi-step production of mRNA, as outlined below. As shown, in a preferred embodiment, the hollow fiber bioreactor (20) comprises a reaction mixture, preferably a DNA template (e.g., linear plasmid, PCR product, amine-functionalized surface-tethered PCR product), It may contain a reaction cell (31) configured to contain an amount of RNA polymerase, and a reaction buffer, as well as other components necessary for the in vitro production of mRNA. A mixing reaction cell (31) may be positioned within the feed chamber (32). This internal mixed reaction cell (31) comprises, in embodiments, a feed solution containing nucleotide NTPs, a reaction buffer which may contain the components necessary to catalyze and drive mRNA synthesis as described herein, and pyrroline It may be in fluid communication with a feed chamber (32) holding pyrophosphatase that catalyzes the hydrolysis of acid to inorganic phosphate. As shown below, in a multi-step system, the feed chamber (32) may contain a feed solution containing enzymes for 5' capping and poly(A) tailing of mRNA transcripts. As described above, the components of the feed solution placed in the feed chamber (32) pass through the porous barrier and into the reaction cell (32) to synthesize mRNA and/or 5' of the mRNA transcripts. A gradient can be formed with the reaction cell (32) to act as a substrate for capping and poly(A) tailing.

In a preferred embodiment, the reaction cell (32) is separated from the feed chamber by a porous barrier (which may preferably be a hollow fiber barrier (34) which may comprise a MWCO PES membrane with pores between the size of 5 kDa or 20 kDa). (32). This fiber membrane can be further treated to reduce protein and nucleotide binding. This treatment involves depositing RNA-free acetylated BSA on the outside of the conduit, and purified RNA polymerase can be placed inside the conduit where mRNA can be produced.

As outlined above, the multi-step in vitro production system (30) of the present invention comprises a multi-step in vitro production system (30) configured to separate mRNA synthesis and poly(A) tailing and, optionally, 5' capping of mRNA transcripts. of bioreactors. In this embodiment, mRNA is produced in a first bioreactor and poly(A) tailing of mRNA transcripts is performed in a second bioreactor. As further outlined below, the 5′ prime capping and modification of the cap may be performed concurrently with mRNA synthesis in the first bioreactor, or separated from the mRNA synthesis step in a second bioreactor. It may be performed during poly(A) tailing.

In the schematic flow diagram provided in Figures 5A-B, the multi-step in vitro production system (30) of the present invention may comprise a first bioreactor, which in this embodiment is the synthesis of mRNA macromolecules. and optionally a first hollow fiber reactor (20a) configured for 5' capping of the transcript. In particular, it is preferred to use the first hollow fiber reactor (20a), but it is not necessary as any in vitro mRNA bioreactor system can be used with the present invention. Referring again to Figures 5A-B, the first hollow fiber reactor (20a) contains a first stage reaction mixture (21), preferably a DNA template (e.g. linear plasmid, PCR product, amine functional a reaction cell (31) configured to contain a surface-tethered PCR product), a first amount of RNA polymerase (SEQ ID NO: 1), and a reaction buffer. This reaction cell (31) of the first hollow fiber reactor (20a) comprises a first stage feed solution (22) containing nucleotide NTPs, which catalyze and drive mRNA synthesis as described or incorporated herein. It may be in fluid communication with a supply chamber (32) holding a reaction buffer, which may contain the necessary components for the reaction, as well as pyrophosphatase, which catalyzes the hydrolysis of pyrophosphate to inorganic phosphate. In this embodiment, the first stage feed solution (22) may be circulated in countercurrent flow as compared to the first stage reaction mixture (21). In this embodiment, the feed chamber (32) and reaction cell (33) are individually or collectively configured for continuous or batch inflow and outflow of components as outlined herein. can be

As described above, the compartmentalization and countercurrent flow of the first stage feed solution (22) and the first stage reaction mixture (21) is such that liberated NT from the first stage feed solution (22) is Passing through the feed chamber (32), it can be drawn into the reaction cell (31) where the RNAP associates with a DNA template having a target sequence encoding the target mRNA (14) and is transferred to the target mRNA nucleotide forming the a of NT. A gradient is generated such that it can react with the components of the first stage reaction mixture (21) so that the incorporation of can be enzymatically catalyzed. This reaction cycle can be carried out for a predetermined period of time (preferably 3-4 hours) or until a certain amount of mRNA is produced in the reaction cell (31). The spent first stage feed solution (23) may be extracted from the feed chamber (32) during or after the cycle is completed.

In this embodiment, the second stage reaction mixture (24) containing mRNA transcripts lacking the poly(A) tail, and the 5′ cap are added to the reaction cell (32) of the first hollow fiber bioreactor (20a). ) and introduced into the mixing cell (33) in which the newly synthesized mRNA is diluted with high salt buffer, preferably at a ratio of 1:5. Dilution and addition of high salt buffer produces a highly concentrated RNA solution, inactivates RNAP, and further adjusts buffer conditions to reduce poly(A) during the second stage poly(A) tailing step. Allows enzymatic activity of the polymerase.

In particular, two major modifications (the 5' cap structure and the poly(A) tail) must be included to produce a mature mRNA primed for efficient translation by the ribosome. The m7G cap structure consists of a 7-methylguanosine triphosphate linked to the 5' end of the mRNA via a 5'→5' triphosphate linkage (m7G cap). The m7G cap (also known as the Cap0 structure) is essential for most protein translation in vivo. The m7G cap also protects mature mRNA from degradation, allows for regulated degradation machinery, enhances pre-RNA splicing, and directs nuclear export. In vivo, the Cap0 structure can be further modified into a Cap1 structure by adding a methyl group to the 2'O position of the starting nucleotide of the mRNA. 2'O methylation in the Cap1 structure helps mRNAs evade innate immune responses in vivo and is particularly important for mRNAs produced for therapeutic use. More specifically, 5' capping is the first step in cotranscriptional pre-mRNA processing, and in many eukaryotes the capping machinery is directly coupled to the phosphorylated C-terminal domain of RNAP. The biosynthesis of the Cap0 structure involves three sequential enzymatic activities: hydrolysis of the 5′ triphosphate terminus of the nascent transcript to a diphosphate by RNA triphosphatase; Requires capping of the diphosphate with GMP by RNA guanylyltransferase using covalently bound GMP as an intermediate and finally methylation of the 5′ guanine base at position N7 by RNA methyltransferase (MT). do.

Referring again to Figures 5A-B, the modified reaction mixture (25) is introduced into the second stage reaction mixture (24) in the mixing cell (33) or, alternatively, the second hollow fiber It can be introduced into the reactor (20b). As noted above, the modification reaction mixture (25) may optionally contain enzymes for 5' capping, as well as specific enzymes for poly(A) tailing of mRNA transcripts. Additional enzymes can be added that initiate polyadenylation and extend the pol(A) tail. The second stage reaction mixture (24) may contain amounts of one or more of the following:
- mRNA triphosphatase enzyme,
- an RNA guanylyltransferase enzyme,
- mRNA methyltransferase enzyme,
- a poly(A) polymerase enzyme,
- a polyadenylation initiation enzyme, and - a polyadenylation elongation enzyme.

In preferred embodiments, the second stage reaction mixture (24) may comprise amounts of one or more of the following:
- an mRNA triphosphatase enzyme according to the amino acid sequence of SEQ ID NO:2,
- an RNA guanylyltransferase enzyme according to the amino acid sequence of SEQ ID NO:3,
- an mRNA capped guanine-N7 methyltransferase enzyme according to the amino acid sequence of SEQ ID NO: 4,
- a poly(A) polymerase enzyme according to the amino acid sequence of SEQ ID NO:5,
- a polyadenylation initiator enzyme according to the amino acid sequence of SEQ ID NO:6, and - a polyadenylation elongation enzyme according to the amino acid sequence of SEQ ID NO:7.

In this configuration, the Cap1-generating methyltransferase (SEQ ID NO:4) recognizes and binds to Cap0-GTP on mRNA transcripts, generating Cap1 methylation. Poly(A) polymerase recognizes and binds Cap1-methyltransferase, forming a complex, and the enzymes act cooperatively as selection tools so that all poly(A)-tailed RNAs are also capped. As such, it begins to generate poly(A) tails. In particular, the five enzymes required for 5' capping of mRNA transcripts are alternatively coupled in the first hollow fiber reactor (20a) for mRNA synthesis and 5' capping, as described below. As such, it may be added to the first stage feed solution (22) and included in the first stage reaction mixture (21) containing the corresponding capping component.

Referring again to Figures 5A-B, the second stage reaction mixture (24) of the above mRNA transcripts is mixed in a dilution buffer in a mixing cell (33) or, alternatively, in a second hollow fiber. A reactor (20b) may also be provided. This new solution is then introduced into a second bioreactor, preferably containing the nucleotide triphosphates (such as ATP and GTP) and S-adenosylmethionine (such as ATP and GTP) required for poly(A) tailing and 5′ capping, respectively. SAM) is introduced into the reaction cell (31) of a second hollow fiber reactor (20b) having a feed chamber (32) containing additional components such as SAM). As noted above, these components pass through a porous barrier, such as the hollow fiber barrier (34) described herein, into the reaction cell (31) to provide 5' capping of mRNA transcripts and poly (A) may be placed in a feed chamber (32) forming a gradient with the reaction cell to act as a substrate for tailing;

The mRNA is capped and poly(A) tailed during one or more cycles of capping and poly(A) tailing. The spent modification reaction mixture (26) can be extracted from the hollow fiber reactor (20b), the mRNA concentrate (27) from the reaction cell (31) of the second hollow fiber reactor (20b) is extracted, A nucleotide removal device (28) can be introduced to remove any free nucleotides to form a purified mRNA (29) output. In this embodiment, the mRNA concentrate (27) from the reaction cell (31) of the second hollow fiber reactor (20b) is diluted in high salt buffer to give poly(A)-tailed RNA poly-dT It may allow slow binding to the resin. Other components pass over the resin and are collected. In certain embodiments, the recombinant enzyme from the reaction mixture is captured by the affinity resin on the column and then washed on the poly dT resin. The reaction mixture was washed thoroughly on poly dT resin, and after the poly(A)-modified RNA was bound to the resin, it was washed with flowing high salt buffer, and after washing, the distilled poly(A)-modified RNA was washed with distilled water. Release from the resin. The final product of the multi-step in vitro production system is concentrated capped and poly(A)-tailed RNA in distilled water (29), which is encapsulated in lipid nanoparticles (LNPs) for therapeutic applications. may be further subjected to additional processing such as

Therefore, in another preferred embodiment, the target mRNA (14) is purified or isolated mRNA. The term "purified mRNA" or "isolated mRNA" as used herein refers to the in vitro should be understood as mRNA having a higher purity after certain purification steps (e.g., alcohol purification and, e.g., HPLC, TFF, and other polynucleotide precipitation steps) than the mRNA transcribed in . Typical impurities that are essentially absent from purified mRNA include peptides or proteins (e.g., enzymes derived from DNA-dependent RNA in vitro transcription (e.g., RNA polymerase, RNase), BSA, pyrophosphatases, restriction endonucleases, DNase), spermidine, immature RNA sequences, RNA fragments, free nucleotides (modified nucleotides, conventional NTPs, Cap analogues), plasmid DNA fragments, buffer components (HEPES, TRIS, MgCI2), and the like. Other impurities (eg, which may result from fermentation procedures) include bacterial impurities (bioburden, bacterial DNA) or impurities from purification procedures (such as organic solvents). Therefore, in this regard, "RNA purity" should be as close to 100% as possible. It is also desirable for RNA purity that the amount of full-length RNA transcript be as close to 100% as possible. Thus, "purified mRNA" or "isolated mRNA" as used herein is greater than 70%, 75%, 80%, 85%, particularly 90%, 91%, 92%, 93% %, 94%, 95%, 96%, 97%, greater than 98%, and most preferably greater than or equal to 99%. Purity can be determined, for example, by analytical HPLC, and the above percentages correspond to the ratio between the area of the target RNA peak and the total area of all peaks representing by-products. Alternatively, purity can be determined by, for example, analytical agarose gel electrophoresis or capillary gel electrophoresis.

The term "dried mRNA" as used herein is understood as mRNA that has been freeze-dried or spray-dried or spray-lyophilized as defined above to obtain a temperature-stable dry mRNA (powder). It must be. "Dried mRNA" as defined herein, and "purified mRNA" as defined herein, or "GMP grade mRNA" as defined herein, have superior stability characteristics and improved efficiency. (eg, better translatability of mRNA in vivo).

In a further embodiment, the invention provides a composition, in a continuous flow bioreactor (1), or in a batch fed reaction if the mRNA encodes an antigenic peptide (particularly a multivalent COVID-19 mRNA vaccine). In vitro transcribed mRNA in the chamber (10) and at least one pharmaceutically acceptable carrier. In particular, the composition according to the invention preferably comprises at least one mRNA as described herein, wherein the spike protein subunit 1 (S1) of the COVID-19 coronavirus, ii) the receptor binding motif of S1 ( RBM), and ii) a nucleocapsid protein (NCP), and fragments or variants thereof, or at least one antigenic peptide or protein comprising or consisting of fragments or variants of any one of these proteins. .

A composition according to the invention is preferably provided as a pharmaceutical composition or vaccine. A "vaccine" is typically understood to be a prophylactic or therapeutic material that provides at least one epitope of an antigen (preferably an immunogen). In some embodiments, the mRNA may encode a peptide that carries or presents at least one epitope. The term "provided on at least an epitope" means, for example, that the vaccine comprises the epitope (or an antigen that contains or presents the epitope), or that the vaccine, for example, the molecule encoding the epitope, or the epitope Containing or providing an antigen is meant. Antigens preferably stimulate the adaptive immune system to provide an adaptive immune response. The (pharmaceutical) compositions or vaccines provided herein further comprise at least one pharmaceutically acceptable excipient, adjuvant or further component (e.g. additive, auxiliary substance etc.) obtain. In a preferred embodiment, the (pharmaceutical) composition or vaccine according to the invention comprises a plurality of mRNAs transcribed in vitro in a continuous flow bioreactor (1) or a batch-fed reaction chamber (10), described herein. may further comprise a multivalent COVID-19 mRNA vaccine administered. According to another embodiment, the (pharmaceutical) composition or vaccine according to the invention may comprise an adjuvant, preferably added to enhance the immunostimulatory properties of the composition. In this context an adjuvant may be understood as any compound suitable to support the administration and delivery of the composition according to the invention. Moreover, such adjuvants can, without being bound by it, initiate or augment the immune response of the innate immune system, ie, a non-specific immune response. In other words, a composition according to the invention, when administered, is typically encoded by at least one coding sequence of the mRNA of the invention contained in the composition of the invention, as defined herein. Initiate an adaptive immune response with an antigen or fragment or variant thereof. In addition, the composition according to the invention is capable of generating a (supportive) innate immune response by adding an adjuvant as defined herein to the composition according to the invention.

The entity of the mRNA vaccine produced by one or more of the methods outlined above, as well as the (pharmaceutical) compositions of the invention, are provided in liquid and/or dried (e.g., lyophilized) form. obtain. They can contain further components, in particular further components which enable their pharmaceutical use. The mRNA, mRNA vaccine or (pharmaceutical) composition thereof may additionally contain, for example, a pharmaceutically acceptable carrier and/or further auxiliary substances and additives and/or adjuvants. The mRNA, mRNA vaccine, or (pharmaceutical) composition thereof, as defined herein, typically comprises a safe and effective amount of the mRNA as defined herein, encodes an antigenic peptide or protein as defined herein, or a fragment or variant thereof, or preferably a combination of antigens as defined herein. As used herein, "therapeutically effective amount" means an amount of mRNA that is sufficient to significantly induce a positive immune response that preferably prevents infection with the COVID-19 coronavirus. At the same time, however, a "therapeutically effective amount" is an amount low enough to avoid serious side effects, ie, to allow a reasonable relationship between benefit and risk. Determination of these limits is typically within the bounds of sound medical judgment. With regard to the mRNA, mRNA vaccine or (pharmaceutical) composition of the invention, the expression "therapeutically effective amount" preferably means the mRNA, mRNA vaccine or (pharmaceutical) composition thereof suitable for stimulating the adaptive immune system. objective) means the amount of the composition, in such a manner that no excessive or detrimental immune response is achieved, but preferably no less than a measurable level of such immune response is achieved. Such a "therapeutically effective amount" of mRNA of a (pharmaceutical) composition or vaccine as defined herein further depends on the type of mRNA, e.g. monocistronic, bicistronic or multicistronic mRNA. can be selected by This is because bicistronic, or even multicistronic, mRNA can result in significantly higher expression of the encoded antigen than using equal amounts of monocistronic mRNA. A "therapeutically effective amount" of the mRNA, mRNA vaccine, or (pharmaceutical) composition of those defined above may further include the specific condition being treated, as well as the age and physical condition of the patient being treated, the severity of the condition. , the duration of treatment, the nature of the accompanying therapy, the particular pharmaceutically acceptable carrier used, and similar factors, and will vary within the knowledge and experience of the attending physician. The mRNA, mRNA vaccine or (pharmaceutical) composition thereof according to the invention can be used according to the invention for humans and for veterinary purposes as a pharmaceutical composition or as a vaccine. .

In a preferred embodiment, the mRNA of the (pharmaceutical) composition, preferably the multivalent COVID-19 mRNA vaccine or kit of parts according to the invention, is provided in lyophilized form. Preferably, the lyophilized mRNA is reconstituted in a suitable buffer prior to administration, advantageously based on an aqueous carrier, such as Ringer's solution, which is preferably lactated Ringer's solution, phosphate buffer. Configured. In a preferred embodiment, the (pharmaceutical) composition, vaccine or kit of parts according to the invention comprises at least 1, 2, 3, 4, 5, 6 or more mRNAs, preferably containing separately provided mRNAs (optionally with at least one further additive) in lyophilized form, preferably so as to allow separate administration of each of the (monocistronic) mRNAs , reconstituted separately in a suitable buffer (eg, Ringer's lactate solution) before their use. A vaccine or (pharmaceutical) composition according to the invention may typically contain a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable carrier" as used herein preferably includes liquid or non-liquid based vaccines of the invention. When the vaccine of the invention is provided in liquid form, the carrier is water, typically pyrogen-free water, isotonic saline, or a buffered (aqueous) solution (e.g., phosphate buffered solution, citrate buffered solution). solution, etc.). In particular, for injection of the vaccines of the invention, water or preferably buffers, more preferably aqueous buffers, can be used, including sodium salts (preferably at least 50 mM sodium salts), calcium salts ( preferably at least 0.01 mM calcium salts), and optionally potassium salts (preferably at least 3 mM potassium salts). According to a preferred embodiment, the sodium, calcium and optionally potassium salts are in the form of their halides (e.g. chlorides, iodides or bromides), their hydroxides, carbonates It can occur in the form of salts, bicarbonates, sulfates, and the like. Examples of sodium salts include, for example, NaCI, Nal, NaBr, a2C(1/4, NaHCCh, _a2S04 ; examples of optional potassium salts include, for example, KCI, KI, KBr, K2CO3, Examples of calcium salts include, but are not limited to, CaCb, Cal2, _CaBr2 , CaCC>3, CaSC, Ca(OH) ₂ . Organic anions may be contained in the buffers.According to a more preferred embodiment, the buffers suitable for injection purposes as defined above are sodium chloride (NaCI), calcium chloride (CaCb) and optionally It may contain a salt selected from potassium chloride (KCI), and in addition to chloride, anion may also be present.CaCb may also be replaced by another salt such as KCI.Typically, injection buffer The salts in the fluid are present at concentrations of at least 50 mM sodium chloride (NaCI), at least 3 mM potassium chloride (KCI), and at least 0.01 mM calcium chloride (CaCb).The injection buffer is a specific reference medium. may be hypertonic, isotonic or hypotonic with respect to, i.e. the buffer may have a higher, the same or a lower salt content with respect to the particular reference medium; can be used, which does not result in cell damage due to osmosis or other concentration effects.Reference media can be blood, lymph, cytosolic fluid, or other body fluids, or, for example, common buffers or liquids such as A medium in an "in vivo" method that occurs in a liquid, such as a liquid that can be used as a reference medium in an "in vitro" method.Such common buffers or liquids are known to those skilled in the art.Lactated Ringer's solution is particularly preferred as the liquid base.

However, one or more compatible solid or compatible liquid fillers or diluents or encapsulating compounds that are suitable for administration to humans may also be used. The term "compatible" as used herein means that a component of the vaccine of the invention will substantially reduce the pharmaceutical efficacy of the vaccine of the invention under typical conditions of use. It means that it can be mixed with the mRNA according to the invention as defined herein in such a way that wax interactions do not occur. Pharmaceutically acceptable carriers, fillers and diluents must, of course, be of sufficiently high purity and sufficiently low toxicity to be suitable for administration to the person being treated. Some examples of compounds that can be used as pharmaceutically acceptable carriers, fillers, or constituents thereof include sugars (such as lactose, glucose, trehalose, and sucrose), starches (such as corn starch or potato starch), dextrose, cellulose and its derivatives (e.g. sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, etc.), powdered tragacanth, malt, gelatin, tallow, solid lubricants (e.g. stearic acid, magnesium stearate, etc.), Calcium sulfate, vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, com oil, and oils derived from cocoa), polyols (such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol), alginic acid.

The choice of pharmaceutically acceptable carrier is in principle determined by the manner in which the pharmaceutical composition or vaccine according to the invention is administered. A composition or vaccine can be administered, for example, systemically or locally. Routes of systemic administration generally include transdermal, oral, parenteral routes, including, for example, subcutaneous, intravenous, intramuscular, intraarterial, intradermal, and intraperitoneal injections, and/or intranasal routes of administration. is included. Routes of local administration generally include, for example, topical routes of administration, but also intradermal, transdermal, subcutaneous, or intramuscular injection, or intralesional, intracranial, intrapulmonary, intracardiac, and sublingual injection. mentioned. More preferably, the composition or vaccine according to the invention may be administered by intradermal, subcutaneous or intramuscular routes, preferably by injection, which may be needle-free injection and/or needle injection. Accordingly, compositions/vaccines are preferably formulated in liquid or solid form. Suitable amounts of vaccines or compositions according to the invention to be administered can be determined by routine experimentation, eg, using animal models. Such models include, without any limitation, rabbit, sheep, mouse, rat, dog, and non-human primate models. Preferred unit dosage forms for injection include sterile solutions of water, saline, or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices. Pharmaceutically acceptable carriers suitable for topical application include those suitable for use in lotions, creams, gels, and the like. For oral administration of the composition or vaccine of the invention, tablets, capsules and the like are preferred unit dosage forms. Pharmaceutically acceptable carriers for the preparation of unit dosage forms that can be used for oral administration are well known in the art. The choice is not critical for the purposes of the present invention and will depend on secondary considerations such as taste, cost, and shelf life, which those skilled in the art have no difficulty making.

The following scientific background information and definitions are provided for clarity and readability. Any technical feature disclosed thereby may be part of each and every embodiment of the present invention. Additional definitions and explanations may be provided in the context of this disclosure.

Poly(A) sequences; poly(A) tails, also referred to as "3'-poly(A) tails or poly(A) sequences," typically add up to about 400 adenosine nucleotides to the 3' end of RNA. long sequences of adenosine nucleotides, such as from about 25 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 is. Additionally, poly(A) sequences or poly(A) tails may be used, for example, in E. It can be generated in vitro by enzymatic polyadenylation of RNA using poly(A) polymerase from E. coli or yeast.

Polyadenylation: Polyadenylation is typically understood to be the addition of poly(A) sequences to a nucleic acid molecule, such as an RNA molecule (eg, immature mRNA). Polyadenylation can be induced by so-called polyadenylation signals. This signal is preferably located within a stretch of nucleotides at the 3' end of a nucleic acid molecule, such as a polyadenylated RNA molecule. Polyadenylation signals typically comprise a hexamer, preferably a hexameric sequence AAUAAA, consisting of adenine and uracil/thymine nucleotides. Other sequences, preferably hexameric sequences, are also conceivable. Polyadenylation typically occurs during processing of pre-mRNA (also called immature mRNA). Typically, RNA maturation (from pre-mRNA to mature mRNA) includes a polyadenylation step.

5' cap structure: 5' caps are typically modified nucleotides (cap analogs), especially guanine nucleotides, added to the 5' end of an mRNA molecule. Preferably, a 5'-5'-triphosphate linkage (also called m7GpppN) is used to add the 5' cap. Further examples of 5' cap structures include glyceryl, inverted deoxy abasic residues (moieties), ',5' methylene nucleotides, l-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbon Cyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seco nucleotides, acyclic 3 , 4-dihydroxybutyl nucleotide, acyclic 3,5′-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediol phosphate, 3′-phosphoroamidate, hexyl phosphate, aminohexyl phosphate, 3′-phosphate, 3′-phosphorothioate, phosphorodithioate, or Crosslinked or non-bridged methylphosphonic acid moieties are included. These modified 5' cap structures can be used in the context of the invention to modify the mRNA sequences of the compositions of the invention. Additional modified 5′ cap structures that may be used in the context of the present invention are CAP1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), CAP2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN). , CAP3 (additional methylation of the ribose at the 3rd nucleotide downstream of m7GpppN), CAP4 (additional methylation of the ribose at the 4th nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphothioate-modified ARCA), inosine, Nl-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azides. - is guanosine.

Vaccine: A vaccine is typically understood to be a prophylactic or therapeutic material that provides at least one antigen or antigenic function. An antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response. An antigen-presenting mRNA in the context of the present invention may typically be an mRNA having at least one open reading frame that can be translated by the cell or organism that provides the mRNA. The product of this translation is a peptide or protein that can act as an antigen, preferably as an immunogen. The product may also be a fusion protein composed of multiple immunogens, e.g. a fusion protein consisting of two or more epitopes, peptides or proteins derived from the same or different viral proteins, wherein the epitopes, peptides or Proteins can be joined by a linker sequence.

Adjuvant component: In its broadest sense, an adjuvant or adjuvant component is typically capable of modifying (e.g. enhancing) the efficacy of drugs or other agents such as vaccines (e.g. pharmacological or immunological chemical) agent or composition. Conventionally, the term, in the context of this invention, refers to compounds or compositions that act as carriers or adjuvant substances for immunogens and/or other pharmaceutically active compounds. This should be interpreted broadly and refers to a wide range of substances capable of increasing the immunogenicity of an antigen incorporated into or co-administered with the adjuvant in question. In the context of the present invention, adjuvants preferably enhance certain immunogenic effects of the active agents of the invention. Typically, "adjuvant" or "adjuvant component" have the same meaning and can be used interchangeably. Adjuvants, for example, can be divided into immunostimulants, antigen delivery systems, or even combinations thereof. In the context of the present invention, adjuvants such as the mRNA vaccines outlined herein and immunostimulatory RNA (isRNA) can be pharmaceutical compositions.

The term "expression" or "expression of a coding sequence" (e.g., gene or transgene), as used herein, means that the encoded information of a nucleic acid transcription unit (e.g., comprising genomic DNA or cDNA) is It often refers to processes that are transformed into an operational, non-operational, or structural part of a cell, including the synthesis of proteins. Gene expression can be influenced by external signals, such as exposing a cell, tissue, or organism to an agent that increases or decreases gene expression. Gene expression can also be regulated anywhere along the pathway from DNA to RNA to protein. Modulation of gene expression is, for example, through regulation affecting transcription, translation, transport and processing of RNA, degradation of intermediate molecules (e.g. mRNA), or activation of specific protein molecules after they are produced. , inactivation, compartmentalization, or degradation, or by a combination thereof. Gene expression at the RNA or protein level by any method known in the art including, but not limited to, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assays. can be measured.

The term "nucleic acid" or "nucleic acid molecule" includes single- and double-stranded forms of DNA, single-stranded forms of RNA, and double-stranded forms of RNA (dsRNA). The terms "nucleotide sequence" or "nucleic acid sequence" refer to both the sense and antisense strands of the nucleic acid either as individual single-stranded or double-stranded strands. The term "ribonucleic acid" (RNA) refers to iRNA (inhibitory RNA), dsRNA (double-stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (microRNA), hpRNA (hairpin RNA). ), tRNA (transfer RNA) (whether charged or discharged with the corresponding acetylated amino acid), cRNA (complementary RNA). The term "deoxyribonucleic acid" (DNA) includes cDNA, genomic DNA, and DNA-RNA hybrids. "Nucleic acid segment" and "nucleotide sequence segment", or more generally the term "segment", may be used to refer to genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller engineered nucleotide sequences ( It will be understood by those of skill in the art as functional terms that include both peptides, polypeptides, or those that encode or can be adapted to encode proteins.

The terms "gene" or "sequence" refer to a coding region operably linked to suitable regulatory sequences capable of regulating the expression of the gene product (eg, polypeptide or functional RNA) in some way. A gene includes the untranslated regulatory regions (e.g. promoters, enhancers, repressors, etc.) of DNA preceding (upstream) and following (downstream) the coding region (open reading frame, ORF) and, if applicable, the individual coding region. It includes intervening sequences (ie, introns) between (ie, exons). As used herein, the term "structural gene" is intended to mean a DNA sequence that is transcribed into mRNA and then translated into a sequence of amino acids characteristic of a particular polypeptide. Note that any reference to a SEQ ID NO or sequence specifically includes that sequence as well as all corresponding sequences corresponding to the first sequence. For example, for any amino acid sequence specified, specifically includes all compatible nucleotide (DNA and RNA) sequences that give rise to that amino acid sequence or protein, and vice versa.

A nucleic acid molecule can comprise either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. A nucleic acid molecule may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those of skill in the art. Such modifications include, for example, labelling, methylation, substitution of one or more analogues of naturally occurring nucleotides, internucleotide modifications such as uncharged bonds such as methylphosphonates, phosphotriesters, phospho loamidates, carbamates, etc., charged linkages: e.g., phosphorothioates, phosphorodithioates, etc., side chain moieties: e.g., peptides, intercalators: e.g., acridine, psoralen, etc., chelating agents; alkylating agents, and modified linkages: e.g. , α anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially double-stranded, triplex, hairpin, circular, and padlock conformations.

In certain embodiments, the invention may encompass in vitro production of artificial and wild-type mRNA. An artificial mRNA (sequence) can typically be understood to be an mRNA molecule that does not occur in nature. In other words, an artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such mRNA molecules are non-naturally occurring due to their individual sequences (which are not naturally occurring) and/or due to other modifications (e.g., non-naturally occurring nucleotide structural modifications). could be. Typically, an artificial mRNA molecule may be designed and/or produced by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). In this context, an artificial sequence is usually a sequence that cannot occur in nature, ie, differs from the wild-type sequence by at least one nucleotide. The term "wild type" can be understood as the naturally occurring sequence. Furthermore, the term "artificial nucleic acid molecule" is not limited to "one molecule", but is typically understood to include an ensemble of identical molecules. Therefore, it may relate to multiple identical molecules contained in an aliquot.

In certain embodiments, the present invention typically comprises bicistronic/multicistronic mRNA: two or more (bicistronic) or (multicistronic) open reading frames (ORFs) (coding regions or coding regions). sequence). An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such an mRNA yields two (bicistronic) or more (multicistronic) different translation products, provided the ORFs are not identical. For expression in eukaryotes, such mRNAs may include, for example, an internal ribosome entry site (IRES) sequence.

In one embodiment, the in vitro produced mRNA is preferably configured to be translated in a host organism (eg, mammalian or human subject in need thereof) to form a peptide. Peptides are polymers of amino acid monomers. Usually the monomers are linked by peptide bonds. The term "peptide" does not limit the length of the polymer chain of amino acids. In some embodiments of the invention, peptides may contain, for example, less than 50 monomeric units. Longer peptides, also called polypeptides, typically have 50-600 monomer units, more particularly 50-300 monomer units.

In one embodiment, the in vitro methods described herein are capable of producing stabilized polynucleotides, preferably stabilized mRNAs that: Stabilized nucleic acids (preferably mRNAs) are typically increases resistance to in vivo degradation (e.g. degradation by exonucleases or endonucleases) and/or ex vivo degradation (e.g. during manufacturing processes prior to vaccine administration, e.g. during preparation of vaccine solutions to be administered) Indicates modification. Stabilization of RNA can be achieved, for example, by providing a 5' cap structure, poly(A) tail, or any other UTR modification. This can also be achieved by chemical modification or modification of the G/C content of the nucleic acid. Various other methods are known in the art and are envisioned in the context of the present invention.

A "pharmaceutical composition" refers to a vaccine of the invention and agents that may typically be used within the pharmaceutical composition or vaccine to facilitate administration of the components of the pharmaceutical composition or vaccine to an individual. (eg, a carrier) and

As used herein, "polymerase" refers to an enzyme that catalyzes the polymerization of nucleotides. A "DNA polymerase" catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase I, T7 DNA polymerase, and Thermus aquaticus (Taq) DNA polymerase. "RNA polymerase" catalyzes the polymerization of ribonucleotides. The DNA polymerases of the preceding examples are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases also fall within the scope of DNA polymerases. Reverse transcriptase is an example of an RNA-dependent DNA polymerase, including viral polymerases encoded by retroviruses. Known examples of RNA polymerases (“RNAPs”) include, among others, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase and E. coli RNA polymerase. The aforementioned examples of RNA polymerases are also known as DNA-dependent RNA polymerases. Polymerase activity of any of the above enzymes can be determined by means well known in the art.

The terms "about" or "approximately" are within a statistically significant range of one or more values such as concentration, length, molecular weight, pH, time frame, temperature, pressure, or volume stated. means that Such values or ranges may be within an order of magnitude of a given value or range, typically within 20%, more typically within 10%, and even more typically within 5%. The allowable variation encompassed by "about" or "approximately" depends on the particular system under study. The terms "comprising," "having," "including," and "containing," unless otherwise noted, are open-ended terms (i.e., "including , but not limited to”).

Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein. Each separate value, including the endpoint boundaries that define the range, is incorporated herein as if it were individually recited herein.

Sequence Listing SEQ ID NO: 1
AA/DNA
RNA polymerase T7 bacteriophage MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTTLLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASA IGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGG GYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNP QGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVN EILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGE ILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPA KGNLNLRDILESDFFAFA
SEQ ID NO:2
amino acid mRNA_triPase
[Insert] ^1
1 Remark: Add organism name from which mRNA trypase is derived YRNVPIWAQKWKPTIKALQSINVKDLKIDPSFLNIIPDDDLTKSVQDWVYATIYSIAPELRSFIELEMKFGVIIDAKGPDRVNPPVSSQCVFTELDAHLTPNIDASLFKELSKYIRGISEVTENTGKFSIIESQTRD SVYRVGLSTQRPRFLRMSTDIKTGRVGQFIEKRHVAQLLLYSPKDSYDVKISLNLELPVPDNDPPEKYKSQSPISERTKDRVSYIHNDSCTRIDITKVENHNQNSKSRQSETTHEVELEINTPALLNAFDNITNDSKEYASLIRTFLNNGT IIRRKLSSLSYEIFEGSKKVM
SEQ ID NO:3
amino acid mRNA guanylyltransferase chlorella virus MVPPTINTGKNITTERAVLTLNGLQIKLHKVGESRDDIVAKMKDLAMDDHKFPRLPGPNPVSIERKDFEKLKQNKYVVSEKTDGIRFMMFFTRVFGFKVCTIIDRAMTVYLLPFKNIPRVLFQGSIF DGELCVDIVEKKFAFVLFDAVVVSGVTVSQMDLASRFFAMKRSLKEFKNVPEDPAILRYKEWIPLEHPTIIKDHLKKANAIYHTDGLIIMSVDEPVIYGRNFNLFKLKPGTHHTIDFIIMSEDGTIGIFDPNLRKNVPVGKLDGYYNKG SIVECGFADGTWKYIQGRSDKNQANDRLTYEKTTLLNIEENITIDELLDLDFKWE
SEQ ID NO: 4
amino acid mRNA cap guanine-N7-methyltransferase Encephalitozoon kunikuli MEGKKEEIREHYNSIRERGRESRQRSKTINIRNANNFIKACLIRLYTKRGDSVLDLGCGKGGDLLKYERAGIGEYYGVDIAEVSINDARVRARNMKRRFKVFFRAQDSYGRHMD LGKEFDVISSQFSFHYAFSTSESLDIAQRNIARHLRPGGYFIMTVPSRDVILERYKQGRMSNDFYKIELEKMEDVPMESVREYRFTLLDSVNNCIEYFVDFTRMVDGFKRLGLSLVERKGFIDFYEDEGRRRNPELSKKMGLGCLTRE ESEVVGIYEVVVFRKLVPE SDA
SEQ ID NO:5
Amino Acid Poly(A) Polymerase Saccharomyces cerevisiae MSSQKVFGITGPVSTVGATAAENKLNDSLIQELKKEGSFETEQETANRVQVLKILQELAQRFVYEVSKKKNMSDGMARDAGKIFTYGSYRLGVHGPGSDIDTLVVVPKHVTREDFFTVFDSLLRERKELD EIAPVPDAFVPIIKIKFSGISSIDLICARLDQPQVPLSLTLSDKNLLRNLDEKDLRALNGTRVTDEILELVPKPNVFRIALRAIKLWAQRRAVYANIFGFPGGVAWAMLVARICQLYPNACSAVILNRFFIILSEWNWPQPVILKPIEDGPLQVRV WNPKIYAQDRSHRMPVITPAYPSMCATHNITESTKKVILQEFVRGVQITNDIFSNKKSWANLFEKNDFFFFRYKFYLEITAYTRGSDEQHLKWSGLVESKVRLLVMKLEVLAGIKIAHPFTKPFESSYCCPTEDDYEMIQDKYGSHK TETALNALKLVTDENKEEESIKDAPKAYLSTMYIGLDFNIENKKEKVDIHIPCTEFVNLCRSFNEDYGDHKVFNLALRFVKGYDLPDEVFDENEKRPSKKSKRKNLE
SEQ ID NO:6
amino acid VP39
Vaccinia virus MDVVSLDKPFMYFEIDNELDYEPESANEVAKKLPYQGQLKLLLGELFFLSKLQRHGILDGATVVYIGSAPGTHIRYLRDHFYNLGVIIKWMLIDGRHHDPILNGLRDVTLVTRFVDEEYLRSIKKQLHPSKIILISDV RSKRGGNEPSTADLLSNYALQNVMISILNPVASSLKWRCPFPDQWIKDFYIPHGNKMLQPFAPSYSAEMRLLSIYTGENMRLTRVTKSDAVNYEKKMYYLNKIVRNKVVNFDYPNQEYDYFHMYFMLRTVYCNKTFPTTKAKV LFLQQSIFRFLNIPTTSTEKVSHEPIQRKISSKNSMSKNRNSKRSVRSNK
SEQ ID NO:7
amino acid VP55
Vaccinia virus MNRNPDQNTLPNITLKIIETYLGRVPSVNEYHMLKLQARNIQKITVFNKDIFVSLVKKNKKRFFSDVNTSASEIKDRILSYFSKQTQTYNIGKLFTIIELQSVLVTTYTDILGVLTIKAPNVISSKISYNVTSEMEEARDMLNSM NVAVIDKAKVMGRHNVSSLVKNVNKLMEEYLRRHNKSCICYGSYSLYLINPNIRYGDIDILQTNSRTFLIDLAFLIKFITGNNIILSKIPYLRNYMVIKDENDNHIIDSFNIRQDTMNVVPKIFIDNIYIVDPTFQLLNMIKMFSQID RLEDLSKDPEKFNARMATMLEYVRYTHGIVFDGKRNNMPMKCIIDENNRIVTVTTKDYFSFKKCLVYLDENVLSSDILDLNADTSCDFESVTNSVYLIHDNIMYTYFSNTILLSDKGKVHEISARGLCAHILLYQMLTSGEYKQCLSD LLNSMMNRDKIPIYSHTERDKKPGRHGFINIEKDIIVF

Claims (46)

A continuous-flow recombination system for producing messenger RNA (mRNA) polynucleotides in vitro, comprising:
- a continuous flow bioreactor,
- at least one continuous flow reaction chamber configured to hold an input reaction mixture with a DNA template; and - configured to hold and circulate a feed solution between said input reaction mixture and said feed solution. at least one continuous flow conduit further configured to be in fluid communication with said continuous flow reaction chamber through a series of conduit openings forming a gradient;
- a continuous flow bioreactor, wherein said input reaction mixture and said feed solution contain all the necessary components for the in vitro production of target mRNA transcribed from said DNA template;
- a protein removal component configured to remove the protein fraction of said mRNA output;
- a DNA removal component configured to remove the DNA fraction of said mRNA output;
- a continuous flow recombination system comprising a nucleotide precipitation component configured to remove the nucleotide triphosphate (NTP) fraction of said mRNA output. 2. The system of claim 1, wherein said DNA template comprises a linear DNA template or a circular DNA template. 3. The system of claim 1 or 2, wherein said DNA template encodes an antigenic polypeptide. 2. The system of claim 1, wherein said antigenic polypeptide comprises an antigenic polypeptide from COVID-19 coronavirus. wherein the input reaction mixture is
- a first amount of an isolated RNA polymerase (RNAP) enzyme,
- an amount of reaction buffer; and - optionally, an initial amount of isolated nucleotide triphosphates (NTPs). System as described. The feed solution is
- a first amount of isolated NTPs,
- an amount of reaction buffer,
- optionally a component of an inorganic polyphosphate energy regeneration system; and - optionally one or more cofactors for said production of mRNA polynucleotides. 3. The system of claim 1, comprising components. 2. The system of claim 1, wherein said protein removal component comprises a protein affinity column configured to remove said protein fraction from said mRNA output. 2. The system of claim 1, wherein said DNA removal component comprises a DNA affinity column configured to remove said protein fraction from said mRNA output. 2. The system of claim 1, wherein said nucleotide precipitation component comprises an alcohol precipitation system configured to isolate said target mRNA from said mRNA output. 10. The system of claim 1 or 9, wherein the isolated target mRNA is resolubilized or dried. 10. The system of claim 9, wherein said isolated target mRNA comprises stabilizing mRNA. 12. The system of claim 9 or 11, wherein said isolated target mRNA is a vaccine. 13. The system of any one of claims 9, 11, 12, wherein said isolated target mRNA is incorporated into a pharmaceutical composition. 3. The system of claim 1, further comprising an input reservoir coupled with an input valve configured to allow real-time injection of feed solution into said continuous flow conduit. 2. The system of claim 1, further comprising an output reservoir coupled with an output valve configured to allow extraction of said mRNA output from said continuous flow reaction chamber. 1. A batch-fed recombinant system for producing messenger RNA (mRNA) polynucleotides in vitro, comprising:
- a batch feeding reaction chamber configured to hold an input reaction mixture comprising a DNA template and a feeding solution, wherein said input reaction mixture and said feeding solution are used for the in vitro production of target mRNA transcribed from said DNA template; a batch-fed reaction chamber containing all the necessary components for
- a protein removal component configured to remove the protein fraction of said mRNA output;
- a DNA removal component configured to remove the DNA fraction of said mRNA output;
- a batch fed recombination system comprising a nucleotide precipitation component configured to remove the nucleotide triphosphate (NTP) fraction of said mRNA output. 17. The system of claim 16, wherein said DNA template comprises a linear DNA template or a circular DNA template. 18. The system of claim 16 or 17, wherein said DNA template encodes an antigenic polypeptide. 17. The system of claim 16, wherein said antigenic polypeptide comprises an antigenic polypeptide from COVID-19 coronavirus. wherein the input reaction mixture is
- a first amount of an isolated RNA polymerase (RNAP) enzyme,
- an amount of reaction buffer; and - optionally, an initial amount of isolated nucleotide triphosphates (NTPs). System as described. The feed solution is
- a first amount of isolated NTPs,
- an amount of reaction buffer,
- optionally a component of an inorganic polyphosphate energy regeneration system; and - optionally one or more cofactors for said production of mRNA polynucleotides. 17. The system of claim 16, comprising components. 17. The system of claim 16, wherein said protein removal component comprises a protein affinity column configured to remove said protein fraction from said mRNA output. 17. The system of claim 16, wherein said DNA removal component comprises a DNA affinity column configured to remove said protein fraction from said mRNA output. 17. The system of claim 16, wherein said nucleotide precipitation component comprises an alcohol precipitation system configured to isolate said target mRNA from said mRNA output. 24. The system of claim 16 or 23, wherein the isolated target mRNA is resolubilized or dried. 24. The system of claim 16 or 23, wherein the isolated target mRNA comprises stabilizing mRNA. 26. The system of any one of claims 16, 23, 25, wherein said isolated target mRNA is a vaccine. 26. The system of claim 23 or 25, wherein said isolated target mRNA is incorporated into a pharmaceutical composition. 17. The system of Claim 16, further comprising an input reservoir coupled with said batch feed reaction chamber. 17. The system of Claim 16, further comprising an output reservoir coupled with said batch feed reaction chamber. A method for continuous flow recombinant production of messenger RNA (mRNA) comprising:
- establishing a continuous flow bioreactor, said continuous flow bioreactor comprising:
- at least one continuous flow reaction chamber configured to hold an input reaction mixture with a DNA template; and - configured to hold a feed solution, wherein a gradient is formed between said input reaction mixture and said feed solution. at least one continuous flow conduit further configured to be in fluid communication with said continuous flow reaction chamber through a series of conduit openings forming;
- establishing that the input reaction mixture and the feed solution contain all the necessary components for the in vitro production of target mRNA transcribed from the DNA template;
- circulating the feed solution through the continuous flow conduit, wherein the constituents of the feed solution pass through the conduit opening where they synthesize the target mRNA directed by the DNA template; circulating interacting with said components of the reaction mixture;
- extracting an mRNA output containing said target mRNA from said continuous flow reaction chamber;
- removing the protein fraction of said mRNA output;
- removing the DNA fraction of said mRNA output;
- removing the nucleotide triphosphate (NTP) fraction of said mRNA output. 31. The method of claim 30, wherein said DNA template comprises a linear DNA template or a circular DNA template. 32. The method of claim 30 or 31, wherein said DNA template encodes an antigenic polypeptide. 31. The method of claim 30, wherein said antigenic polypeptide comprises an antigenic polypeptide from COVID-19 coronavirus. wherein the input reaction mixture is
- a first amount of an isolated RNA polymerase (RNAP) enzyme,
- an amount of reaction buffer; and - optionally, an initial amount of isolated nucleotide triphosphates (NTPs). described method. The feed solution is
- a first amount of isolated NTPs,
- an amount of reaction buffer,
- optionally a component of an inorganic polyphosphate energy regeneration system; and - optionally one or more cofactors for said production of mRNA polynucleotides. 31. The method of claim 30, comprising a component. said step of removing said protein fraction of said mRNA output, wherein said step of removing said protein fraction of said mRNA output is configured to remove said protein fraction from said mRNA output; a protein affinity column; 31. The method of claim 30, comprising: said step of removing said DNA fraction of said mRNA output, wherein said step of removing said DNA fraction of said mRNA output is configured to remove said DNA fraction from said mRNA output; a DNA affinity column; 31. The method of claim 30, comprising: 31. The method of claim 30, wherein said step of removing said NTP fraction of said mRNA output comprises said step of precipitating said NTP fraction from said mRNA output. 39. The method of claim 30 or 38, further comprising resolubilizing or drying said isolated target mRNA. 39. The method of claim 30 or 38, wherein the isolated target mRNA comprises stabilizing mRNA. 41. The method of claim 38 or 40, wherein said isolated target mRNA is a vaccine. 42. The method of any one of claims 38, 40, 41, further comprising incorporating said isolated target mRNA into a pharmaceutical composition. 31. The method of claim 30, further comprising infusing said feed solution from an input reservoir into said continuous flow conduit. 31. The method of claim 30, further comprising extracting mRNA output from said continuous flow reaction chamber into an output reservoir. 31. The method of claim 30, wherein said output reservoir comprises a DNA extraction component, a protein extraction component, or an NTP extraction component.

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